Periconceptional Non-medical Maternal Determinants Influence the Tryptophan Metabolism: The Rotterdam Periconceptional Cohort (Predict Study)

Background: The vital role of the maternal tryptophan (TRP) metabolism in maternal health and pregnancy is well established. However, non-medical maternal determinants influencing the TRP metabolism have been poorly investigated. We hypothesise that periconceptional maternal non-medical determinants alter the TRP metabolism, affecting both kynurenine (KP) and serotonin pathway (SP) metabolite concentrations. Therefore, we investigated the influence of non-medical maternal determinants on the TRP metabolism during the periconception period. Methods: About 1916 pregnancies were included from the Rotterdam Periconceptional Cohort between November 2010 and December 2020. Data on periconceptional non-medical maternal determinants were collected through questionnaires. Serum samples were collected at 8.5 (SD = 1.6) weeks of gestation and TRP, kynurenine (KYN), 5-hydroxytryptophan (5-HTP), 5-HT (5-hydroxytryptamine) and 5-hydroxyindole acetic acid (5-HIAA) were determined using validated liquid chromatography (tandem) mass spectrometry. Mixed models were used to determine associations between periconceptional non-medical maternal determinants and these metabolites. Results: In total 11 periconceptional non-medical maternal determinants were identified. Protein intake was positively associated with TRP (β = .12, 95% CI = 0.07-0.17), while age, energy intake and body mass index (BMI) (β = −.24, 95% CI = −0.37 to −0.10) were negatively associated with TRP. Age, BMI and total homocysteine were associated with higher KYN, whereas non-western geographical origin was associated with lower KYN (β = −.09, 95% CI = −0.16 to −0.03). Protein intake and total homocysteine (β = .07, 95% CI = 0.03-0.11) had a positive association with 5-HTP, while a negative association was found for energy intake. A non-western geographical origin and drug use were associated with higher 5-HT, and BMI with lower 5-HT (β = −6.32, 95% CI = −10.26 to −2.38). Age was positively associated with 5-HIAA (β = .92, 95% CI = 0.29-1.56), and BMI negatively. Conclusions: Periconceptional non-medical maternal determinants, including age, geographical origin, drug use, energy and protein intake, BMI and total homocysteine, influence KP and SP metabolite concentrations.


Introduction
Tryptophan is an essential amino acid required for protein synthesis, and is the precursor of many bioactive metabolites.Tryptophan is mainly metabolised along the kynurenine pathway (KP) in the liver (>95%) and the serotonin pathway (SP) in the gut and brain (Figure 1). 1,2][4] SP metabolites are important for brain and gut function and are involved in the stress response. 5,6The effects of tryptophan metabolites can be both beneficial and harmful depending on the concentration and the cell type.A balanced tryptophan metabolism is important to maintain homoeostasis in cells and tissues.Indeed, alterations of the tryptophan metabolism have been associated with various non-communicable diseases, such as cancer, cardiovascular, and psychiatric diseases.][9] Several non-communicable diseases originate during the periconception period, a critical period covering the 14 weeks before and the 10 weeks after conception.It involves essential metabolic processes that programme the developing embryo through epigenetic processes, with potential long-term health implications. 10Maternal exposures, such as poor lifestyle behaviours, including malnutrition, during this period are 2 International Journal of Tryptophan Research known to disturb several metabolic pathways, including the one-carbon metabolism. 104][15][16][17][18] Derangement of the one-carbon metabolism often coincides with disruption of the tryptophan metabolism, as the tryptophan metabolism provides once-carbon units for the folate cycle and shares cofactors with the one-carbon metabolism. 10,19Tryptophan metabolites are involved in redox reactions, and can have proand anti-oxidative properties.Disruption of the tryptophan metabolism can induce excessive oxidative stress and subsequent inflammation, which are involved in adverse pregnancy outcomes. 20,21A body of evidence suggests that changes of maternal tryptophan metabolism, resulting in either an increase or decrease of the KP or SP, contribute to adverse pregnancy outcomes.Specifically, lower maternal tryptophan concentrations have been associated with depression during pregnancy, gestational diabetes, foetal growth restriction, and preterm birth, while higher kynurenine concentrations have been associated with gestational diabetes, and preterm birth. 22dditionally, there is evidence indicating that maternal tryptophan can modulate foetal brain development through placental synthesis of serotonin. 23However, our study specifically focussed on maternal tryptophan metabolite concentrations, and thus did not encompass an analysis of placental tryptophan metabolite concentrations.
Since the periconception period is a critical window of exposure, which shapes women's and offspring's health, increasing our understanding of maternal determinants that can influence the maternal tryptophan metabolism during the periconception period is of interest with regards to future prevention and early interventions of pregnancy complications and non-communicable diseases later in life.The influence of medical determinants on the tryptophan metabolism is widely acknowledged, especially concerning inflammatory diseases and medication use. 24,25In contrast, there are only a few studies that have assessed non-medical maternal determinants in relation to the tryptophan metabolism, with a particular scarcity of studies conducted during the periconception period.In this context, non-medical determinants refer to non-modifiable determinants such as age and geographical origin, as well as modifiable determinants such as lifestyle behaviours, and educational level.We postulate that periconceptional non-medical maternal determinants modify the tryptophan metabolism, leading to alterations of KP and SP metabolite concentrations.Therefore, the aim of this explorative study was to identify non-medical maternal determinants that increase or decrease KP and SP metabolite concentrations during the periconception period.The analytes determined in this study are coloured grey.Tryptophan is converted to N-formylkynurenine by the hepatic tryptophan 2,3-dioxygenase (TDO) or the extrahepatic indole amine 2,3-dioxygenase (IDO).This is the rate-limiting step in the kynurenine pathway.N-formylkynurenine is hydrolysed to kynurenine by arylformamidase.Kynurenine is further metabolised into the downstream kynurenine pathway metabolites kynurenic acid, anthranilic acid, 3-hydroxy-anthranilic acid, quinolinic acid, picolinic acid, and nicotinamide adenine dinucleotide (NAD+).Tryptophan is converted to 5-hydroxytryptophan, which is the rate limiting step of the serotonin pathway.Then, 5-hydroxytryptophan is converted to 5-hydroxytryptamine by aromatic amino acid decarboxylase.Finally, 5-hydroxytryptamine is converted to 5-hydroxyindoleacetic acid via 2 enzymatic steps involving the enzymes monoamine oxidase and aldehyde dehydrogenase.
van Zundert et al 3

Study design and setting
This study was embedded within the Rotterdam Periconceptional Cohort (Predict Study), an ongoing prospective tertiary hospital-based cohort study performed at the Department of Obstetrics and Gynaecology of the Erasmus MC, University Medical Centre, the Netherlands (Erasmus MC). 26,27The Predict Study was conducted in accordance with the Declaration of Helsinki and approved by the Central Committee on Research in The Hague and the local Medical Ethics Committee of the Erasmus MC (15 October 2004,  MEC-2004-277).Prior to inclusion, all participants provided written informed consent. 26,27Women and their partners were eligible to participate in the Predict Study if they had a child wish, and were at least 18 years old, as well as proficient in speaking and reading Dutch. 26,27

Study population
The current study focussed exclusively on women included from November 2010 to December 2020, that is, 2051 women with an average age of 32.4 (range: 18.0-48.6)years.Since this study focussed on tryptophan metabolites in serum collected during the first trimester of pregnancy, women were excluded from this study if they were not willing to provide a blood sample during this period.This resulted in a total study population of 1916 pregnancies.

Non-medical determinants
The periconceptional non-medical maternal determinants investigated in this study were classified into 3 categories: nonmodifiable determinants, modifiable lifestyle determinants, and other modifiable determinants (Table 1).Data on these determinants were collected using a general questionnaire and a validated food frequency questionnaire (FFQ) covering the periconception period, which were filled out before the intake appointment at the hospital.7][28][29] Maternal age was calculated at the moment of conception.Geographical origin was defined as non-Western or Western. 30Smoking, alcohol use, and drug use covered any substance used during the periconception period.Details regarding specific drugs were inquired, encompassing marijuana, amphetamine, XTC, cocaine, methadone, heroin, and unspecified other drugs.Energy intake (kilojoules) and protein intake (grams) were calculated per day from the FFQ.Folic acid supplement use was considered adequate if initiated before conception.Educational level was classified into low, middle and high based on the International Standard Classification of Education (ISCED). 31ody mass index (BMI) was calculated by the dividing the individual's weight (kilograms) by the square of their height (metres).Total homocysteine (µmol/L) was measured in serum at 8.5 (SD = 1.6) gestational weeks as marker for lifestyle behaviours, including nutrition. 10

Statistical methods
Baseline characteristics were presented as means (standard deviation (SD)) or median (interquartile range (IQR)) for continuous variables depending on their distribution and as number of individuals (percentages) for categorical variables.Based on visual inspection of the distribution, the tryptophan metabolites were considered approximately normally distributed, except for 5-hydroxyindole acetic acid.Therefore, 5-hydroxyindole acetic acid was (natural) log transformed in all analyses.For the tryptophan metabolites a scatter plot matrix was created.
Mixed models were used to determine associations between periconceptional non-medical maternal determinants and tryptophan metabolites to account for possible correlations between pregnancies of the same women.Bivariable analyses

International Journal of Tryptophan Research
were conducted for each determinant and tryptophan metabolite.Furthermore, a multivariable analysis was performed by including all determinants in a single model.The analysis was adjusted for gestational age at the blood draw.
To assess the robustness of the findings a cubic spline function was used to detect non-linear associations between the determinants and the outcomes and all two-way interaction terms were added to the multivariable model.Both did not improve the fit of the model, as indicated by higher or comparable Akaike information criterion (AIC) and Bayesian information criterion (BIC).
All statistical analysis were performed using R version 4.2.1. 33The results were presented as effect estimates with 95% confidence intervals (95% CI).A P ⩽ .05 was considered statistically significant.

Main results
Supplemental Table 1 presents the results from the bivariable model and Table 3 from the multivariable model.The results of the multivariable analysis are described below and displayed in Figure 3.All associations mentioned in the Results section were statistically significant, unless otherwise specified.
Non-modifiable determinants.Age was negatively associated with TRP and positively with KYN and 5-HIAA (TRP:  Modifiable lifestyle determinants.Figure 3 shows a clear decreasing trend of all tryptophan metabolites in women who smoked.No consistent trend was found with alcohol use.Drug use was strongly associated with 5-HT (β = 174.23,95% CI = 38.34-310.12,P = .012),but not with other tryptophan metabolites.
Albeit not statistically significant, a positive association was found between folic acid supplement use and TRP and SP metabolites and a negative association with KYN.
Other modifiable determinants.Highly educated women had increased TRP and SP metabolite concentrations, while women with a low educational level had decreased 5-HTP and 5-HIAA concentrations compared to women with a medium educational level.However, none of these associations were statistically significant.

Summary of results
This study demonstrates that non-medical maternal determinants affected the tryptophan metabolism during the periconception period, as shown by increased and decreased KP and SP metabolite concentrations (Figure 4).The most pronounced and statistically significant associations are summarised in this paragraph.Age at conception was negatively associated with TRP and positively with KYN and 5-HIAA.A negative association was found between a non-western geographical origin and KYN, while a positive association was found with 5-HT.Drug use was strongly positively associated with 5-HT.Energy intake was negatively associated with TRP and 5-HTP, whereas protein intake was positively associated with TRP and 5-HTP.For BMI and total homocysteine, a positive association was found with KYN and with 5-HTP only for total homocysteine.BMI was negatively associated with TRP and SP metabolites.
Given the distinctions observed in tryptophan metabolism across species in earlier research, we prioritised human studies as our primary comparative reference. 37,38In instances where human data were lacking, we utilised relevant data from animal studies.

Non-modifiable determinants
0][41][42][43][44][45] Ageing is associated with an increasing pro-inflammatory status.This can cause upregulation of indole amine 2,3-dioxygenase (IDO) (Figure 1), leading to decreased TRP and increased KYN concentrations. 41,46A proposed mechanism for the age-related increase of 5-HIAA, is a compensatory response to the diminished efficiency of transport mechanisms with increasing age. 44ur findings regarding the association between a non-western geographical origin and tryptophan metabolites are in accordance with 2 earlier much smaller studies reporting decreased KYN and increased 5-HT concentrations in Afro-Americans, Hispanic-Americans and Asians compared to Caucasians. 47,48The mechanisms that explain the changes of tryptophan metabolites between different geographical origins are still largely unclear, but could be related to variations in genetics, diets and gut microbiota. 49

Modifiable lifestyle determinants
Any smoking during the periconception period was negatively associated with KP and SP metabolites in our study, which is consistent with prior research on (chronic) smoking. 39,50onversely, one small study (n = 35) found a positive association between smoking and 5-HT measured 15 minutes after smoking a cigarette. 51This is in line with prior studies in rats, which reported increased 5-HT concentrations after acute  .785 The multivariable model included all non-medical determinants and was additionally adjusted for gestational age at the blood draw.Statistical significant results (P ≤ .05)are bold.
administration of nicotine and decreased 5-HT concentrations after chronic administration of nicotine. 52An inhibitory effect of smoking on IDO has been reported and subsequent decreases KYN concentrations. 53,54Even though, it is suggested that IDO activity is reduced by smoking, no increased TRP concentrations were found in the present study or in previous studies. 53,54urprisingly, the present study showed no associations between any periconceptional alcohol use and tryptophan metabolites, possibly attributed to the relatively low levels of alcohol use among these women during the periconception period.Previous research on the effect of alcohol on tryptophan metabolites has mainly focussed on chronic alcohol use and demonstrated increased KYN concentrations via activation of tryptophan 2,3-dioxygenase (TDO). 55vidence suggests that drug use can alter both the KP as the SP of the tryptophan metabolism. 55,56We found a strong positive association between any periconceptional drug use and 5-HT, but considering the small number of women who used drugs and the different types of drugs used, this result should be interpreted with caution.The most reported drug in our study was cannabis, which can potentially influence the serotonin synthesis by inhibiting IDO.This can result in increased availability of TRP for the SP. 57otal homocysteine was positively associated with KYN, which substantiates the positive correlation between total homocysteine and the KYN/TRP ratio observed in earlier studies. 41Previous literature has demonstrated that folate, as substrate in the one-carbon metabolism, and the tryptophan metabolism are intertwined, as the tryptophan metabolism supplies one-carbon units to the folate metabolism. 10This is demonstrated by the FIGLU test, a diagnostic tool for folate deficiency used in the past, which is also positive when tryptophan deficiency is present.The conversion of FIGLU into glutamic acid is facilitated by the coenzyme tetrahydrofolate, a derivate of folate which is reliant on tryptophan as one of the one-carbon sources. 10,58Folate deficiency can lead to derangements in the one-carbon metabolism, of which total homocysteine is a sensitive biomarker. 10Hyperhomocysteinaemia and alterations of tryptophan metabolite concentrations are both involved in oxidative and inflammatory pathways. 10,20,21n addition, a deficiency in shared cofactors and substrates essential for both the tryptophan metabolism and one-carbon metabolism may potentially explain the observed positive associations between total homocysteine, KYN, and 5-HTP concentrations.Vitamin B6 serves as an illustrative example as it is crucial not only for converting total homocysteine into cystathionine, but also for the conversion of KYN into downstream metabolites and for the conversion of 5-HTP to 5-HT. 10,19,59,60However, future research is warranted to elucidate the exact mechanism underlying the observed associations, and explore the role of various cofactors in this complex interplay between the tryptophan metabolism and one-carbon metabolism.The opposite effect of periconceptional energy intake and periconceptional protein intake on TRP and SP metabolites is in agreement with a prior study including only nine participants. 61When digested from protein rich food, the essential amino acid TRP is taken up in the circulation.It is suggested that in response to an energy-rich meal insulin increases, which supports protein synthesis by facilitating amino acids into the cells, resulting in decreased TRP concentrations. 61,62

Other modifiable determinants
To the best of our knowledge, no previous studies have assessed the association between educational level as exposure and tryptophan metabolite concentrations as outcomes.The negative association of BMI with TRP and SP metabolites and the positive association with KYN in our study are in line with previous studies. 32,63Both increasing BMI and a lower level of education have been associated with chronic inflammation. 64,65Increased pro-inflammatory cytokines can upregulate IDO.Consequently, tryptophan may be directed towards the KP, leaving less tryptophan available for the SP. 2,63

Strengths and limitations
A major strength is the prospective observational study design, which allowed for comprehensive data collection on multiple periconceptional non-medical determinants using (validated) questionnaires. 28Furthermore, the sample size in this study is very large in comparison to previous studies, and noteworthy, particularly considering the complex process of determining tryptophan metabolites.Another strength is the use of an accurate and robust LC-MS/MS method to measure tryptophan metabolites from both the KP and SP, which has been validated in women in the first trimester of pregnancy. 32In fact, this is the first study reporting the absolute ↑, positive association; ↓, negative association, •, no association.The associations that were significant in the multivariable model are coloured black.If the direction of the associations was the same in the bivariable as in the multivariable model but not statistically significant the arrows are coloured grey.Abbreviations: 5-HTP, 5-hydroxytryptophan; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindole acetic acid; KYN, kynurenine; TRP, tryptophan.

International Journal of Tryptophan Research
concentrations of tryptophan metabolites from both the KP and SP of almost 2000 women in the first trimester of pregnancy.
In the current study, total tryptophan was measured instead of free tryptophan, which is known to be readily available for het KP and SP.However, free tryptophan is difficult to determine and easily influenced by several external determinants, such as timing of the blood sampling, which were not optimal in our study. 66Moreover, 5-HT was only measured in serum, which may have affected the results by 5-HT leakage from platelets.Data on platelet count and 5-HT concentrations in other blood fractions, such as platelet-rich plasma, could have given more information about the 5-HT metabolism and is recommended for future research. 67Despite the interconnectedness of the tryptophan metabolites within the same biochemical pathway, they were analysed individually due to their weak correlations and distinctive functions.Inherent to the explorative character of this study, no corrections for multiple testing have been applied.To further validate our findings and explore additional non-medical determinants, our study should be repeated in a population-based periconceptional cohort study.

Implications and future perspectives
The results of this study suggest that the maternal tryptophan metabolism could be a shared pathway underlying the associations between periconceptional non-medical maternal determinants and adverse health outcomes, including pregnancy complications.][70] Proposed biological pathways include excessive inflammation and oxidative stress. 46,71This study demonstrated that as maternal age and BMI increase, tryptophan concentrations decrease, while kynurenine concentrations increase.][73] Gaining more knowledge of determinants that affect the tryptophan metabolism during the periconception period, allows for a broader perspective on how this metabolism affects multiple aspects of maternal and foetal health.Consequently, this offers opportunities for preventive and therapeutic interventions that target multiple health outcomes simultaneously.Moreover, early markers may be identified that indicate the onset of adverse maternal health outcomes or pregnancy complications, even before clinically relevant and costly health impairments occur.
To gain more insight into the activity of the KP, the LC-MS/ MS method should be expanded by other downstream KP metabolites, and kynurenic acid and quinolinic acid in particular considering their associations with various psychiatric disorders. 74,75However, it is important to consider the challenges in LC-MS/MS method development associated with these downstream metabolites, such as analyte stability, sensitivity and selectivity.Additionally, investigating additional biological pathways that modulate the tryptophan metabolism, such as the ability of cortisol to upregulate IDO in the KP, could shed light on their potential role in the development of pregnancy complications.Understanding the role of tryptophan in the maternal-placental-foetal metabolism, can enhance our knowledge of the aetiology of pregnancy complications, warranting further investigation in future studies.

Conclusion
This study is the first to identify multiple periconceptional non-medical maternal determinants influencing tryptophan metabolism along the KP and SP.Specifically, maternal age, geographical origin, drug use, energy intake, protein intake, BMI, and total homocysteine level during the periconception period were found to be associated with changes in tryptophan metabolites.The maternal tryptophan metabolism may serve as a common pathway linking periconceptional non-medical maternal determinants to adverse pregnancy outcomes.Notably, modifiable non-medical maternal determinants, may be potential preconceptional targets for preventing alterations in tryptophan metabolism, with significant implications for maternal health and pregnancy outcome.

Figure 1 .
Figure 1.Kynurenine and serotonin pathway of the tryptophan metabolism.

Figure 3 .
Figure 3. Forrest plots demonstrating the effect estimates including 95% confidence intervals of the multivariable mixed models for each determinant.

Figure 4 .
Figure 4. Summary of periconceptional non-medical maternal determinants of the tryptophan metabolism.

Table 1 .
Periconceptional non-medical maternal determinants investigated in this study.

Table 2 .
Periconceptional non-medical maternal determinants and tryptophan metabolites of the total study population.

Table 3 .
Adjusted associations between periconceptional non-medical maternal determinants and tryptophan metabolite concentrations in the first trimester of pregnancy.